Synthetic methods for the preparation of indolylquinones and mono- and bis-indolylquinones prepared therefrom

ABSTRACT

The present invention relates to novel synthetic methods for the preparation of indolylquinones. The methods of the present invention are directed to synthetic reactions involving indoles and halo-quinones in solvent and in the presence of a metal carbonate. The invention also relates to bis- and mono-indolylquinones of high purity and pharmaceutical compositions containing the same.

This is a division, of application Ser. No. 08/964,791, filed Nov. 5,1997 now U.S. Pat. No. 5,786,488 which claims the benefit of U.S.Provisional Application No. 60/030,604 filed Nov. 13, 1996 and U.SProvisional Application No. 60/042,989 filed Apr. 14, 1997.

1. INTRODUCTION

The present invention relates to novel synthetic methods for thepreparation of both known and novel indolylquinones. Manyindolylquinones, in particular the class of indolylquinones known as theasterriquinones, have utility in the treatment of cell proliferativedisorders such as cancer. In addition, many indolylquinones are known tobe useful as dyes. Finally, indolylquinones are also known to exhibitantifungal and antibacterial properties. However, in general,indolylquinones are isolated from natural sources and are not preparedsynthetically. The present invention is directed to a synthetic methodfor the preparation of indolylquinones, and to novel compounds preparedusing said method. In particular, the methods of the present inventionare directed to synthetic reactions involving indoles and halo-quinonesin solvent and in the presence of a metal carbonate. These methodsprovide a direct and simple means of preparing the compounds ofinterest, indolylquinones.

2. BACKGROUND OP THE INVENTION

2.1 Isolation From Natural Sources and Therapeutic Utility ofIndolylquinones

Research interest concerning indolylquinones grew out of earlyobservations that extracts of Chaetomium exhibited antibioticproperties. These observations led researchers to attempt the isolationof active species from cultures of these microorganisms. For example,Brewer et al. disclose the isolation of a purple pigment, which wastermed cochliodinol, from isolates of Chaetomium cochliodes andChaetomium globosum (1968, "The Production of Cochliodinol and a RelatedMetabolite by Chaetomium Species," Can. J. Microbiol. 14:861-866).Brewer et al. also disclose the synthetic conversion of cochliodinol toa diacetate compound. Id. Further, the antifungal properties ofcochliodinol have also been documented (Meiler et al., 1971, "The Effectof Cochliodinol, a Metabolite of Chaetomium cochliodes on theRespiration of Microspores of Fusarium oxysporum," Can. J. Microbiol.17: 83-86).

The structure of cochliodinol was elucidated by Jerram et al. in 1975.(1975, "The Chemistry of Cochliodinol, a Metabolite of Chaetomium spp.,"Can. J. Chem. 53:727-737). Jerram et al. reported the structure ofcochliodinol as: 2,5-dihydroxy-3,6-di(5'-(2"-methylbut-Δ²"-ene)-indolyl-3')-cyclohexadiene-1,4-dione. The conversion ofcochliodinol to various other derivatives, including its dimethyl anddiacetyl analogues, was also disclosed. Id. Some of these derivativeswere highly colored and suitable for use as dyes, while others werecolorless. Id. Sekita discloses the isolation of otherbis(3-indolyl)-dihydroxybenzoquinones, including isocochliodinol andneocochliodinol from Chaetomium muroum and C. amygdalisporum (1983,"Isocochliodinol and Neocochliodinol, Bis(indolyl)-benzoquinones fromChaetomium spp.," Chem. Pharm. Bull. 31(9): 2998-3001).

Despite the therapeutic potential of cochliodinol and its derivatives,efficient methods suitable for large scale production of these compoundshave remained elusive. U.S. Pat. No. 3,917,820 to Brewer et al.discloses the purple pigment cochliodinol and a process for itsproduction by culturing various types of Chaetomium under aerobicconditions. However, the methods of Brewer require long incubationperiods for cochliodinol production (2-8 days), the use of benzene, aknown carcinogen, to effect chromatographic separation of cochliodinolfrom the culture and are limited to the few naturally occurringcompounds. Moreover, Brewer discloses the isolation of only smallquantities (0.75 grams) of cochliodinol from Chaetomium.

Another class of indolylquinones known as the asterriquinones in whichthe nitrogen of the indole ring is substituted, has been shown toexhibit antitumor activity. Arai et al. proposed the general name"asterriquinones" for the class of indolylquinones based uponasterriquinone (1981, "Metabolic Products of Aspergillus terreus IV.Metabolites of the Strain IFO 8835. (2) The Isolation and ChemicalStructure of Indolyl Benzoquinone Pigments," Chem. Pharm. Bull. 29(4):961-969). It should be noted that as used herein, the term"asterriquinone" has a more general meaning, and is used interchangeablywith the term "indolylquinone." Yamamoto et.al. disclose the antitumoractivity of asterriquinone, i.e.,2,5-bis[N-(1",1"-dimethyl-2"-propenyl)indol-3'-yl]-3,6-dihydroxy-1,4-benzoquinone,and its isolation from the fungus Aspergillus terreus (1976, "AntitumorActivity of Asterriquinone, a Metabolic Product from Aspergillusterreus," Gann 67:623-624).

Arai et al. disclose the isolation and characterization of 11 differentkinds of bisindolyl-dimethoxyl-p-benzoquinones from Aspergillus terreus.Id. The isolation and structural determination of a number of otherasterriquinones have also been reported. (Arai et al. 1981, "MetabolicProducts of Aspergillus terreus VI. Metabolites of the Strain IFO 8835.(3) the Isolation and Chemical Structures of Colorless Metabolites,"Chem. Pharm. Bull. 29(4): 1005-1012; Kaji et al., 1994, "Four NewMetabolites of Aspergillus Terreus", Chem. Pharm. Bull. 42(8):1682-1684). However, the separation of asterriquinones is troublesomebecause there are so many kinds of homologous pigments in theAspergillus extracts. Moreover, the chromatographic purification ofasterriquinones is typically carried out using benzene, a knowncarcinogen, as a solvent. Finally, only milligram quantities ofasterriquinones have actually been isolated from these natural sources.

In view of their potential as anticancer agents, research has beendirected to determination of the relationship between structure andantitumor activity of asterriquinones. For example, Arai et al. reporteda study in which hydroxyl benzoquinone derivatives obtained bydemethylation of bisindolyl-dimethoxyl-p-benzoquinones were found tohave greater antitumor activity than the methoxyl derivatives (1981,"Metabolic Products of Aspergillus terreus V. Demethylation ofAsterriquinones," Chem. Pharm. Bull. 29(4): 991-999). Shimizu et al.noted that the presence of free hydroxyl groups in the benzoquinonemoiety, as well the number and position of tert-, isopentenyl, or bothpentyl groups, seems to have an effect on the antitumor activity of thecompound (1982, "Antitumor Effect and Structure-Activity Relationship ofAsterriquinone Analogs," Gann 73: 642-648). In an attempt to obtaininformation towards the development of more potent asterriquinonederivatives, Shimizu et al. conducted an investigation into thestructure-activity relationship of asterriquinones in which the actionmechanism of asterriquinone in its antitumor activity with reference toits interaction with DNA molecules and the plasma membrane of tumorcells was studied (1990, "Interaction of Asterriquinone withDeoxyribonucleic Acid in Vitro," Chem. Pharm. Bull. 38(9): 2617-2619).It was reported that a correlation exists between the pKa value of theasterriquinone derivative and its antitumor activity. Id. Maximumantitumor activity was observed for compounds with pKa's in the range of6-7. Id.

Analysis of structure-activity relationships has led to attempts toobtain compounds with more potent antitumor activity by chemicalmodification of asterriquinone and related compounds isolated fromnatural sources (Shimizu et al., 1982, "Antitumor Activity ofAsterriquinones from Aspergillus Fungil IV. An Attempt to Modify theStructure of Asterriquinones to Increase the Activity," Chem. Pharm.Bull. 30(5): 1896-1899). Although benzoquinone derivatives havingaziridinyl groups in the molecule such as mitomycin C, carbazilquinoneor "E 39" are well known potent anticancer agents, replacement of thefunctional groups at the 3 and 6 positions in the benzoquinone moiety ofasterriquinone failed to enhance its antitumor potency. Id. Similarly,the introduction of an ethyleneimino group into the molecule did notincrease antitumor activity. A dimethylallyl derivative ofasterriquinone showed moderate activity against the ascites and solidtumors of Ehrlich carcinoma, while an allyl derivative did not. It wassuggested that in order to enhance the antitumor activity, it may benecessary not only to alter the pKa value by alkylation, but also tointroduce hydrophilic groups into the molecule.

Most recently, in addition to their demonstrated antitumor activity,asterriquinone and some of its analogues have also been shown to bestrong inhibitors of HIV-reverse transcriptase (Ono et al., 1991,"Inhibition of HIV-Reverse Transcriptase Activity by Asterriquinone andits Analogues," Biochem. Biophys. Res. Commun. 174(1): 56-62).

2.2 Cancer and Signal Transduction

As mentioned above, indolylquinones have utility as antitumor agents forthe treatment of cancer and other cell proliferative disorders. Thesecompounds are believed to arrest the growth of tumors by interferingwith the signal transduction pathways that regulate cell proliferationand differentiation.

Protein phosphorylation is a common regulatory mechanism used by cellsto selectively modify proteins carrying signals that regulate cellproliferation and differentiation. The proteins that execute thesebiochemical modifications are a group of enzymes known as proteinkinases. They may further be defined by the amino acid that they targetfor phosphorylation. One group of protein kinases are the tyrosinekinases (PTKs) which selectively phosphorylate a target protein on itstyrosine residues.

Protein tyrosine kinases comprise a large family of proteins, includingmany growth factor receptors and potential oncogenes. Tyrosine kinasescan be cytoplasmic, non-receptor-type enzymes and act as a key componentof a signal transduction pathway which regulates cell functions such ascell division, differentiation and survival.

Adaptor proteins are intracellular proteins having characteristicconserved peptide domains (SH2 and/or SH3 domains, as described below)which are critical to the signal transduction pathway. Such adaptorproteins serve to link protein tyrosine kinases, especiallyreceptor-type protein tyrosine kinases to downstream intracellularsignalling path-ways such as the RAS signalling pathway. It is thoughtthat such adaptor proteins may be involved in targeting signaltransduction proteins to the correct site in the plasma membrane orsubcellular compartments, and may also be involved in the regulation ofprotein movement within the cell.

The profound cellular effects mediated by tyrosine kinases and adaptormolecules have made them attractive targets for the development of newtherapeutic molecules. It is known, for example, that the overexpressionof tyrosine kinases, such as HER2, can play a decisive role in thedevelopment of cancer (Slamon, D. J., et al. , 1987, Science,235:177-182) and that antibodies capable of blocking the activity ofthis enzyme can abrogate tumor growth. (Drebin, et al. 1988, Oncogene2:387-394). Blocking the signal transduction capability of tyrosinekinases such as Flk-1 and the PDGF receptor have been shown to blocktumor growth in animal models (Millauer, B., et al. 1994, Nature367:577; Ueno, H., et al. 1991, Science 252:844-848).

Despite great interest in the various therapeutic and other utilities ofindolylquinones such as asterriquinones, research into the therapeuticactivities of indolylquinones and efforts to obtain indolylquinones withenhanced therapeutic activity have both been limited by the lack ofreliable sources for these compounds. Indeed, isolation ofindolylquinones from natural sources requires multiple steps andproduces only milligram quantities of the target molecules. Further,evaluation of the activities of novel indolylquinones has necessarilybeen confined to those compounds which can be obtained by chemicalmodification of known compounds that can be isolated from naturalsources. Clearly, a synthetic routine to these compounds would beinvaluable to the art.

2.3 Synthesis of Cochliodinol

A synthetic route to an indolylquinone, cochliodinol, has been reportedby Horcher et al. This route is a complex, multi-step, low-yield processfor the total synthesis of cochliodinol (1986, "Totalsynthese desCochliodinols", Liebigs. Ann. Chem. 1765-1771). The Horcher methodinvolves an unusual solid state reaction of bromanil(2,3,5,6-tetrabromo-1,4-quinone) with 5-bromoindole in the presence ofaluminum oxide and potassium carbonate in a dry box at 105° C. Thissolid state reaction yields about 11% of2,5-dibromo-5,6-bis(5-bromo-3-indolyl)-1,4-quinone. The2,5-dibromo-5,6-bis(5-bromo-3-indolyl)-1,4-quinone is then treated withbenzalcohol and sodium hydroxide to give2,5-bis(benzyloxy)-3,6-bis(5-bromo-3-indolyl)-1,4-quinone in 45% yield.This product is then reacted with hydrogen gas in the presence of a 10%Pd on activated charcoal catalyst, followed by treatment with aceticanhydride in pyridine to give1,2,4,5-tetracetoxy-3,6-bis(5-bromo-3-indolyl)benzene. Reaction of thiscompound with a complex of isopentenyl bromide and tetracarbonyl nickelgives1,2,4,5-tetracetoxy-3,6-bis[5-(3-methyl-2-butenyl)3-indolyl]benzene.This compound is then reacted with sodium hydroxide and oxygen to givecochliodinol.

According to Horcher et al., the reaction of bromanil with certainsubstituted indoles is problematic. Horcher et al. report that earlierattempts to react a p-benzoquinone with 2-methylindole resulted in onlymonoindolequinones in very low yields. Attempts to react bromanil with5-(2-methylbut-2-en-4-yl)-indole were also reported by Horcher to beunsuccessful due to the instability of the unsaturated side chainvis-a-vis the dehydrogenating bromanil. To overcome this difficulty,Horcher reacted bromanil with 5-bromoindole instead of5-(2-methylbut-2-en-4-yl), followed by introduction of the2-methylbut-2-en-4-yl group at the end of the synthesis, requiring theadditional step of reacting the 5,5'-dibromo-bis-indolylquinone with thecomplex of isopentenyl bromide and tetracarbonyl nickel, whichsubstitutes the bromine atoms with 2-methylbut-2-en-4-yl groups.

Horcher et al. report that this method resulted in isolation of onlymilligram quantities of cochliodinol in a very low overall yield.However, Horcher et al. indicate that conducting the initial reaction ofbromanil with 5-bromoindole in smaller batches results in better yields.This suggests that the methods of Horcher et al. are unsuited forproduction of bis-indolylquinones on a large scale. In addition, asapplied to the production of bis-indolylquinones in general, the methodsof Horcher et al. would be prohibitively multistep, and would likelyresult in isolation of only milligram quantities of the targetindolylquinones. Moreover, these methods require high temperature andmanipulation in dry box.

Accordingly, despite the great interest in indolylquinones, there is alack of feasible large scale synthetic routes for obtaining thesecompounds. Thus, there is a need in the art for a fast, efficientsynthetic method for making indolylquinones in preparative quantities.Further, there is a need for synthetic means of producing knownindolylquinones previously available only in milligram quantities fromnatural sources. Moreover, there is a need in the art for a syntheticmethod that may be manipulated easily to produce a wide variety ofstructurally diverse novel indolylquinones, so that structure-activityrelationships may be further elucidated, and new, perhaps moretherapeutically useful indolylquinones may be developed.

3. SUMMARY OF THE INVENTION

The present invention provides a method for the synthesis ofindolylquinones which comprise reacting a substituted or unsubstituted2,5-dihalo-1,4-benzoquinone with one or more substituted orunsubstituted indoles in a polar organic solvent and in the presence ofmetal carbonate.

In one embodiment, the present invention provides a method for preparinga symmetrical indolylquinone compound of the formula I: ##STR1##wherein: R1 and R2 are each independently Br, Cl, F, I, H, OH or --OCOR,wherein R is, lower alkyl, aryl or alkylaryl; R"1 is H, C₁ -C₇ alkyl, C₂-C₇ alkenyl, C₂ -C₇ alkynyl, arylalkyl or aryl; and R3 to R7 are eachindependently hydrogen, branched or unbranched C₁ -C_(n) alkyl,alkylcarboxy, C₂ -C_(m) alkenyl, C₂ -C_(m) alkynyl, alkenylcarboxy,aryl, alkylaryl, hydroxy, hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo,trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido,amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from0 to 12, preferably 1-7, and m is an integer from 0 to 12, preferably1-7. R1 and R2 are preferably Br, Cl, F or H; and least preferably OH.The method comprises reacting a substituted or unsubstituted2,5-dihalo-1,4-benzoquinone, preferably a 2,5-dibromo-1,4-benzoquinonecompound of the formula II: ##STR2## wherein R1 and R2 are as definedabove; with at least one indole of the formula III: ##STR3## wherein R"1and R3-R7 are as defined above. The reaction is carried out in a polarorganic solvent and in the presence of metal carbonate under mildconditions which are further discussed below.

The method may further comprise reacting the indolylquinone compound offormula I with an alkali metal hydroxide to produce a compound of theformula IV: ##STR4## Further, the invention encompasses reacting theindolylquinone compound of formula I wherein R1 and R2 are Br with analkali metal hydroxide and an alcohol of the formula R'OH, wherein R' islower alkyl or alkylaryl, to produce an indolylquinone compound of theformula: ##STR5## wherein R'1 and R'2 are each independently loweralkyl, aryl or alkylaryl.

In another embodiment, the present invention provides a method forpreparing an asymmetrical indolylquinone compound of the formula VI:##STR6## wherein: R1 and R2 are each independently Br, Cl, F, I, H, OH,or --OCOR, wherein R is lower alkyl, aryl or alkylaryl; R"1 and R"2 areeach independently H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂ -C₇ alkynyl,arylalkyl or aryl; and R3 to R12 are each independently hydrogen,branched or unbranched C₁ -C_(n) alkyl, alkylcarboxy, C₂ -C_(m) alkenyl,C₂ -C_(m) alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy,hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo, trihalomethyl, amido,carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or2-methylbut-2-en-4-yl, wherein n is an integer from 0 to 12, preferably1-7, and m is an integer from 0 to 12, preferably 1-7. R1 and R2 arepreferably Br, Cl, F or H; and least referably OH. The principal stepsof the method comprise:

(a) reacting a substituted or unsubstituted 2,5-dibromo-1,4-benzoquinonecompound of the formula II: ##STR7## wherein R1 and R2 are as definedabove; with one equivalent of a first indole of the formula III:##STR8## wherein R"1 and R3-R7 are as defined above; in a polar organicsolvent and in the presence of metal carbonate;

(b) reacting the intermediate product of step (a) with one equivalent ofa second indole of the formula IV: ##STR9## wherein R"2 and R8-R12 areas defined above. Both reactions are carried out in a polar organicsolvent and in the presence of metal carbonate under mild conditionswhich are further discussed below.

The invention also encompasses further reacting the indolylquinonecompound of formula VI with an alkali metal hydroxide to produce acompound of the formula VIII: ##STR10## wherein R"1, R"2 and R3-R12 areas defined above.

Further, the invention encompasses reacting the indolylquinone offormula VI wherein R1 and R2 are Br, F, Cl or I with an alkali metalhydroxide and an alcohol of the formula R'OH, wherein R' is lower alkylor alkylaryl, to produce an indolylquinone compound of the formula IX:##STR11## wherein R'1 and R'2 are each independently lower alkyl, arylor alkylaryl.

In yet another embodiment, the present invention provides a method forpreparing an indolylquinone compound of the formula X: ##STR12##wherein: R"1 is H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂ -C₇ alkynyl,arylalkyl or aryl4 and R3 to R7 are each independently hydrogen,branched or unbranched C₁ -C_(n) alkyl, alkylcarboxy, C₂ -C_(m) alkenyl,C₂ -C_(m) alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy,hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo, trihalomethyl, amido,carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or2-methylbut-2-en-4-yl,

wherein n is an integer of 0 to 12, preferably 1-7, and m is an integerof 0 to 12, preferably 1-7. This embodiment of the inventive methodinvolves reacting 2,3,5,6-tetrabromo-1,4-benzoquinone with at least oneindole of the formula: ##STR13## wherein R"1 and R3-R7 are as definedabove. The reaction is carried out in a polar organic solvent in thepresence of metal carbonate.

The method also encompasses further reacting the indolylquinone compoundof formula X with an alkali metal hydroxide to produce a compound of theformula: ##STR14## wherein R3-R7 are as defined above. Further, theinvention also encompasses a method which further comprises reacting theindolylquinone compound of formula X with an alkali metal hydroxide andan alcohol of the formula R'OH, wherein R' is lower alkyl or alkylaryl,to produce an indolylquinone compound of the formula: ##STR15## whereinR'1 and R'2 are each independently lower alkyl, aryl or alkylaryl.

In yet another embodiment, the present invention provides a method forpreparing an indolylquinone compound of the formula XIV: ##STR16##wherein: R"1 and R"2 are each independently H, C₁ -C₇ alkyl, C₂ -C₇alkenyl, C₂ -C₇ alkynyl, arylalkyl or aryl; and R3 to R12 are eachindependently hydrogen, branched or unbranched C₁ -C_(n) alkyl,alkylcarboxy, C₂ -C_(m) alkenyl, C₂ -C_(m) alkynyl, alkenylcarboxy,aryl, alkylaryl, hydroxy, hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo,trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido,amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer of 0to 12, preferably 1-7, and m is an integer of 0 to 12, preferably 1-7.This embodiment of the inventive method involves reacting2,3,5,6-tetrabromo-1,4-benzoquinone with one equivalent of a firstindole of the formula III: ##STR17## wherein R"1 and R3-R7 are asdefined above; in a polar organic solvent and in the presence of metalcarbonate;

reacting the intermediate product of step (a) with one equivalent of asecond indole of the formula VII: ##STR18## wherein R"2 and R8-R12 areas defined above. Both reactions are carried out in a polar organicsolvent and in the presence of metal carbonate under mild conditionswhich are further discussed below.

The invention also encompasses further reacting the indolylquinonecompound of formula XIV with an alkali metal hydroxide to produce acompound of the formula VIII: ##STR19## wherein R"1, R"2 and R3-R12 areas defined above.

Further, the invention encompasses reacting the indolylquinone offormula VI wherein R1 and R2 are Br, F, Cl or I with an alkali metalhydroxide and an alcohol of the formula R'OH, wherein R' is lower alkylor alkylaryl, to produce an indolylquinone compound of the formula IX:##STR20## wherein R'1 and R'2 are each independently lower alkyl, arylor alkylaryl.

In still another embodiment, the present invention provides a method forpreparing a mono-indolylquinone compound of the formula XI: ##STR21##wherein R1, R2 and R30 are each independently Br, Cl, F, I, HI OH or--OCOR, wherein R is lower alkyl, aryl or alkylaryl;

R"1 is H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂ -C₇ alkynyl, arylalkyl oraryl; and

R3 to R7 are each independently hydrogen, branched or unbranched C₁-C_(n) alkyl, alkylcarboxy, C₂ -C_(m) alkenyl, alkynyl, alkenylcarboxy,aryl, alkylaryl, hydroxy, hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo,trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido,amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from0 to 12 and m is an integer from 0 to 12. This method comprises reactinga substituted or unsubstituted 2,5-dibromo-1,4-benzoquinone compound ofthe formula: ##STR22## wherein R1 and R2 are as defined above, with oneindole of the formula ##STR23## wherein R"1 and R3-R7 are as definedabove. The reaction is carried out in a polar organic solvent and in thepresence of metal carbonate.

The method of the present invention may further comprise reacting theindolylquinone compound of formula XI with an alkali metal hydroxide toproduce a compound of the formula: ##STR24## wherein R30 and R3-R7 areas defined above.

The method may further comprise reacting the indolylquinone compound offormula XI wherein R1, R2 and R30 are Br, F, Cl or I with a mixture ofan alkali metal hydroxide and an alcohol of the formula R'OH, wherein R'is lower alkyl or alkylaryl, to produce an indolylquinone compound ofthe formula: ##STR25## wherein R'1 and R'2 are each independently loweralkyl, aryl or alkylaryl.

In another embodiment, the present invention further encompasses methodsfor producing large quantities of known, naturally occurringasterriquinones in high purity and in high yield. In yet anotherembodiment, the present invention is directed to known, syntheticallyprepared naturally occurring asterriquinones of high purity which areobtainable in large quantities and in high yield. The invention alsoencompasses the preparation of novel monoindolylquinones, i.e.,compounds substituted with only one indole, and the imonoindolylquinonecompounds, as described below.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

4. DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention relates to a synthetic routefor the preparation of a wide variety of indolylquinones includingasterriquinone compounds having interesting antitumor activity. As aresult, the invention provides a major contribution to the art in thatnaturally occurring compounds with important therapeutic properties canbe prepared in large quantities, e.g., quantities of about 1 gram ormore, with purity in excess of about 95% and in high yield. Theavailability of large quantities allows the skilled artisan to morequickly and easily test these naturally occurring compounds. Prior tothe present invention, such compounds were isolated from natural sourcesin limited quantities (e.g., about 750 mg or less) and low purity (e.g.,less than about 95%). With the present invention, large quantities canmore easily be obtained and the purification of complex natural productmixtures is avoided. Finally, the present invention provides a means forpreparing novel analogues of the naturally occurring indolylquinones,which analogues may prove to be of greater interest than the naturallyoccurring compounds for their therapeutic activity or other properties.

More specifically, the present invention provides a solvent basedreaction of indoles and haloquinones in the presence of metal carbonate.It has been discovered that the reaction of indoles and halo-quinonesusing a polar organic solvent and metal carbonate provides a rapid andefficient reaction under mild conditions, which include but are notlimited to mild temperatures, short reaction times and standard/ambientpressures. Thus, the present method is well suited for large scale andcommercial production of indolylquinones.

More specifically, the present invention provides methods for thepreparation of indolylquinones which involve the reaction of a2,5-dihalo-1,4-benzoquinone of the formula II with at least one indoleof the formula III. When one indole is used, a symmetricalbis-indolylquinone of the formula I is obtained as the product. In thealternative, step-wise or concurrent addition of two different indolesmay be used to obtain an asymmetrical bis-indolylquinone of the formulaVI. For example, an asymmetrical bis-indolylquinone may be made by thereaction of a 2,5-dihalo-1,4-benzoquinone with about one equivalent of afirst indole, followed by addition of about 1.5 equivalents of a secondindole. Preferably, the preparation of asymmetrical bis-indolylquinonesis carried out in the presence of about 3 equivalents of metalcarbonate. As another alternative, mixtures of two or more indoles maybe reacted with the starting quinone to give a mixture of symmetricaland asymmetrical bis-indolylquinones. Finally, the controlled additionof one equivalent of at least one indole to the starting quinone in thepresence of about one equivalent of metal carbonate can be used toobtain one or more mono-indolylquinones.

The preferred 2,5-dihalo-1,4-quinones useful in the present inventionare the 2,5-dibromo-1,4-benzoquinones of formula II, which may besubstituted or unsubstituted, wherein R1 and R2 are each independentlyBr, Cl, F, I, OH, H or --OCOR, wherein R is lower alkyl, aryl oralkylaryl. Alternatively, the 2,5-dichloro-, difluoro- ordiiodo-1,4-benzoquinones may be used in the methods of the invention. Aparticularly preferred 2,5-dibromo-1,4-quinone is2,3,5,6-tetrabromo-1,4-benzoquinone.

The indoles useful in the present invention may be substituted at the 1,2, 3, 4, 5 or 7 positions with hydrogen, branched or unbranched C₁-C_(n) alkyl, alkylcarboxy, C₂ -C_(m) alkenyl, C₂ -C_(m) alkynyl,alkenylcarboxy, aryl, akylaryl, hydroxy, hydroxyalkyl, C₁ -C_(n) alkoxy,nitro, halo, trihalomethyl, amido, carboxamido, carboxy, sulfonyl,sulfonamido, amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is aninteger from 0 to 12, preferably 1-7, and m is an integer from 0 to 12,preferably 1-7. Preferably, the indoles used in the present inventionare substituted at the 2 position. Least preferred indoles are theunsubstituted indoles. Certain indoles useful in the present inventionare available from commercial sources such as the Aldrich ChemicalCompany, Milwaukee, Wis. Alternatively, the indoles may be prepared viaa cyclization procedure according to the method of Verley and Bedure,1925, Bull. Soc. Chim. Fr. (37): 190.

Any non-nucleophilic, aprotic solvent may be used in the 35 methods ofthe invention. Mixtures of solvents may also be used. Preferred solventsare inert or non-reactive, polar organic solvents including but notlimited to acetonitrile, dimethyl formamide (DMF) and tetrahydrofuran(THF). A particularly preferred solvent is acetonitrile. The solventvolume will depend upon the scale of the reactors, and may range from afew milliliters up to a multi-liter volumes useful in large-scaleproduction. Reactant concentrations are set forth below.

It is believed that the metal carbonate used in the methods of thepresent invention assists the reaction of the 2,5-dihalo-1,4-quinone andthe indole by scavenging the hydrogen bromide by-product formed in thereaction. Any metal carbonate or mixture of metal carbonates may beused; however, cesium carbonate, potassium carbonate, sodium carbonate,lithium carbonate and mixtures thereof are preferred. A particularlypreferred metal carbonate is cesium carbonate. The amount of metalcarbonate used in the method ranges from about 2 to about 10 equivalentsbased on the haloquinone; preferably 2 to 5 equivalents; and mostpreferably 3 to 4 equivalents.

The reaction may be run at any concentration ranging from about 0.1M toabout 5M (molarity based on the haloquinone). Preferably the reaction isrun at a concentration of about 1M.

As mentioned above, the reaction to produce a mono-indolylquinone ispreferably carried out in the presence of about two equivalents of metalcarbonate. Reactions to produce symmetrical or asymmetricalbis-indolylquinones are preferably carried out in the presence of about3 equivalents of metal carbonate.

According to the methods of the invention, the reaction of the2,5-dihalo-1,4-quinone with the indole may be run at temperaturesranging from about -10° C. to about 100° C. However, a particularlybeneficial aspect of the present invention is that harsh conditions andhigh temperatures are not required to effect this reaction. Preferablythe reaction of the 2,5-dihalo-1,4-quinone is run at a temperature inthe range of about 0° C. to about 30° C. More preferably, the reactionis run at about room temperature.

The reaction of the 2,5-dihalo-1,4-quinone with the indole may beconducted under an inert atmosphere such as nitrogen or argon; however,the reaction may also be run in atmospheric air. The reaction may be runat any pressure up to 500 psig; however, it is preferable to conduct thereaction at atmospheric pressure.

The reaction time will vary according to the specific, reactants andreaction conditions used, but generally will be from about 2 hours toabout 72 hours.

After reaction of the 2,5-dihalo-1,4-quinone and the indole, the productindolylquinones are typically isolated according to standard workupprocedures. For example, the crude reaction mixture may be diluted with1N hydrochloric acid, followed by extraction with an organic solventsuch as ethyl acetate. Typically, the organic layer is washed with brineand then dried over anhydrous sodium sulfate. As an alternative toextraction, the crude reaction mixture may simply be filtered to removesolids. The solvent is removed under reduced pressure, and the cruderesidue is purified by recrystallization, flash chromatography, HighPressure Liquid Chromatography (HPLC) or a combination thereof.Preferably, the residue is purified using flash chromatography and/orHigh Pressure Liquid chromatography (HPLC).

In a preferred embodiment, the symmetrical bis-indolylquinone of formulaI is further reacted with an alkali metal hydroxide to give abis-indol-2,5-dihydroxy-1,4-quinone of formula IV. Preferred alkalimetal hydroxides are sodium and potassium hydroxide, or mixturesthereof. Preferably, this reaction is carried out in a mixture ofethanol and tetrahydrofuran using concentrated aqueous KOH at a refluxtemperature of about 85° C. for up to 20 hours. Thebis-indol-2,5-dihydroxy-1,4-quinone of formula IV maybe isolatedaccording to standard workup and purification procedures as describedabove.

In other embodiments, the methods of the present invention comprisereacting a symmetrical compound of formula I wherein R1 and R2 are Br,or an asymmetrical compound of formula VI wherein R1 and R2 are Br, withan alkali metal hydroxide and an alcohol of the formula R'OH wherein R'is lower alkyl or alkylaryl, to a produce symmetrical indolylquinone ofthe formula V or an asymmetrical indolylquinone of formula XI.

As used herein the term "substituted or unsubstituted" means that thegroup in question can be substituted with one or more substituents asdesired; for example, substituents other than hydrogen such as Br, Cl,F, I, NH₂, NR2, NO₂, CN, COR, OH, OR, SO₂, alkyl, aryl, alkylaryl andthe like may be used. On the contrary, unsubstituted groups do notcontain any substituents.

By the term "alkyl" as used herein is meant a straight or branched chainsaturated hydrocarbon group having from 1 to 12 carbons such as methyl,ethyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl, isoamyl, n-hexyl,n-octyl and n-decyl; "alkenyl" and "alkynyl" are used to mean straightor branched chain hydrocarbon groups having from 2 to 12 carbons andunsaturated by a double or triple bond respectively, such as vinyl,allyl, propargyl, 1-methylvinyl, but-1-enyl, but-2-enyl, but-2-ynyl, 1methylbut-2-enyl, pent-1-enyl, pent-3-enyl, 3-methylbut-1-ynyl,1,1-dimethylallyl, hex-2-enyl and 1-methyl-1-ethylallyl; "alkylaryl"means the aforementioned alkyl groups substituted by a phenyl group suchas benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl and2-benzylpropyl; "aryl" as used herein includes a monocyclic or bicyclicrings, wherein at least one ring is aromatic including aromatic orhetero-aromatic hydrocarbons; the term "hydroxy-alkyl" means theaforementioned alkyl groups substituted by a single hydroxyl group suchas 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl,1-hydroxybutyl and 6-hydroxyhexyl.

Specific compounds which can be made according to the methods of thepresent invention are described by formula (XII) below. R1-R12 of theformula can be as listed in Table I following the formula. Illustrativepreparations of these compounds are found in the working examples.

                                      TABLE I                                     __________________________________________________________________________                                                XII 26##                             -                                                                          Ex.                                                                             Compound #                                                                          R1 = R2                                                                            R11       R12       R3-R10.sup.1                                 __________________________________________________________________________     1.                                                                              (1)  H    3-methyl-n-butyl                                                                        3-methyl-n-butyl                                          2.  (1) H 3-methyl-n-butyl 3-methyl-n-butyl                                   3.  (2) H n-butyl n-butyl                                                     4.  (3) H methyl methyl                                                       5.  (4) H 2-methylbut-2-en-4-yl 2-methylbut-2-en-4-yl                         6.  (5) Ac 2-methylbut-2-en-4-yl 2-methylbut-2-en-4-yl                        7.  (6) Ac 3-methyl-n-butyl 3-methyl-n-butyl                                  8.  (1) H 3-methyl-n-butyl 3-methyl-n-butyl                                   9.  (7) H H H R5 = R9 = Br                                                   10.  (8) H allyl allyl                                                        11.  (9) H n-propyl n-propyl                                                  12. (10) H aminocarbonyl aminocarbonyl                                        13. (11) Ac aminocarbonyl aminocarbonyl                                       14. (12) benzoyl allyl allyl                                                  15. (13) H cyano cyano                                                        16. (14) H H H R6 = R10 =                                                          methoxycarbonyl                                                          17. (15) H H H R3 = R5 = R7 = R9 =                                                 methoxy                                                                  18. (16) H H H R3 = R6 = R7 = R10 =                                                methoxy                                                                  19. (17) H H H R5 = R9 = nitro                                                20. (18) H H H R6 = R10 =                                                          4-chlorobenzoylamino                                                     21. (19) H 4-chlorophenyl 4-chlorophenyl                                      22. (20) H 4-fluorophenyl 4-fluorophenyl                                      23. (21) H H H R4 = R6 = R8 = R10 =                                                methoxy                                                                  24. (22) H H H R4 = R5 = R8 = R9 =                                                 methoxy                                                                  25. (23) H H H R6 = R10 = cyano                                               26. (24) H H H R5 = R9 = trifluoro-                                                methylphenyl-                                                                 aminocarbonyl                                                            27. (25) H 4-trifluoromethyl 4-trifluoromethyl                                   phenylaminocarbonyl phenylaminocarbonyl                                    28. (26) H ethyl ethyl                                                        29. (27) H H H R4 = R8 = NO.sub.2                                                  R5 = R9 = Br                                                             30. (28) Me 2-methylbut-2-en-4-yl 2-methylbut-2-en-4-yl                       31. (29) Me 3-methyl-n-butyl 3-methyl-n-butyl                                 32.  (1) H 3-methyl-n-butyl 3-methyl-n-butyl                                  33.  (3) H methyl methyl                                                      34. (26) H ethyl ethyl                                                        35.  (2) H n-butyl n-butyl                                                    36. (31) H but-1-en-4-yl but-1-en-4-yl                                        37. (32) H 4-methyl-n-pentyl 4-methyl-n-pentyl                                38. (33) H 2-phenylethyl 2-phenylethyl                                        39. (34) H H 3-methyl-n-butyl                                                 40. (35) H ethyl ethyl R5 = R9 = carboxy                                      41. (36) H n-propyl n-propyl R5 = R9 = carboxy                                42. (37) H 3-methyl-n-butyl 3-methyl-n-butyl R5 = R9 = carboxy                43. (38) H 4-carboxy-n-butyl 4-carboxy-n-butyl                                44. (39) H H 3-methyl-n-butyl R5 = carboxy                                    45. (40) H ethyl ethyl R5 = R9 = amino                                        46. (41) H n-propyl n-propyl R5 = R9 = amino                                  47. (42) H 3-methyl-n-butyl 3-methyl-n-butyl R5 = R9 = amino                  48.  (6) acetyl 3-methyl-n-butyl 3-methyl-n-butyl                             49. (43) H ethyl ethyl R5 = R9 =                                                   4-methylphenyl-                                                               sulfonylamino                                                            50. (44) H n-propyl n-propyl R5 = R9 =                                             4-methylphenyl-                                                               sulfonylamino                                                            51. (45) H 3-methyl-n-butyl 3-methyl-n-butyl R5 = R9 =                             4-methylphenyl-                                                               sulfonylamino                                                            52. (46) H 2-methylbut-1-en-4-yl 2-methylbut-1-en-4-yl                        53. (47) H 2-methylpent-2-en-5-yl 2-methylpent-2-en-5-yl                      54. (48) H phenyl phenyl                                                      55. (49) H carboxy carboxy                                                    56. (50) H methyl carboxy                                                     57. (51) H methyl phenyl                                                      58. (52) H 3-methyl-n-butyl phenyl                                            59. (53) H n-butyl carboxy                                                    60. (54) H n-propyl carboxy                                                   61. (55) H n-propyl n-propyl R4 = R8 = carboxy                              __________________________________________________________________________     .sup.1 Unless otherwise indicated, R3-R10 = hydrogen.                    

The mono-indolylquinone compounds which can be made according to themethods of the present invention are described by formula (XI) below.R1-R7 and R30 can be as listed in Table II below. ##STR27##

The following is a general experimental procedure for the synthesis ofthe trihalo-monoindolyl quinones of formula XI using the appropriateindoles. As discussed above, the indoles may be commercially availableor may be prepared according to the method of Verley and Bedure, 1925,Bull. Soc. Chim. Fr. (37): 190.

Preparation of 6-(2-phenylindol-3-yl)-2,3,5-tribromo-1,4-quinone[Compound (48a)]: To a 25 ml round bottom flask equipped with a magneticstir bar and a drying tube was placed 2-phenylindole (2.28 g), cesiumcarbonate (7.69 g), bromanil (5 g), and acetonitrile (11.8 ml). Afterstirring the mixture at room temperature for 3 hours, 1N hydrochloricacid (150 ml) was added and the aqueous layer extracted with ethylacetate (300 ml). The organic layer was washed with brine (150 ml) anddried with sodium sulfate. Following removal of the solvent underreduced pressure, the crude residue was purified by flash chromatography(10% ethyl acetate/hexane) to yield6-(2-phenylindol-3-yl)-2,3,5-tribromo-1,4-quinone (1.88 g, 30%) as ablue crystalline solid.

                                      TABLE II                                    __________________________________________________________________________    Compound                                                                        # R1 = R2 = R30 R7 R3-R6.sup.2                                              __________________________________________________________________________    (1a)  Br     3-methyl-n-butyl                                                   (2a) Br n-butyl                                                               (3a) Br methyl                                                                (4a) Br 2-methylbut-2-en-4-yl                                                 (1a) Br 3-methyl-n-butyl                                                      (7a) Br H R5 = Br                                                             (8a) Br allyl                                                                 (9a) Br n-propyl                                                              (10a) Br aminocarbonyl                                                        (13a) Br cyano                                                                (14) Br H R6 = methoxycarbonyl                                                (15a) Br H R3 = R6 = methoxy                                                  (17a) Br H R5 = nitro                                                         (18a) Br H R6 = 4-chlorobenzoylamino                                          (19a) Br 4-chlorophenyl                                                       (20a) Br 4-fluorophenyl                                                       (21a) Br H R4 = R6 = methoxy                                                  (22a) Br H R4 = R5 = methoxy                                                  (23a) Br H R6 = cyano                                                         (24a) Br H R5 = trifluoro-                                                       methylphenylaminocarbonyl                                                  (25a) Br 4-trifluoromethyl-                                                     phenylaminocarbonyl                                                         (26a) Br ethyl                                                                (27a) Br H R4 = NO.sub.2 ; R5 = Br                                            (1a) Br 3-methyl-n-butyl                                                      (31a) Br but-l-en-4-yl                                                        (32a) Br 4-methyl-n-pentyl                                                    (33a) Br 2-phenylethyl                                                        (34a) Br H                                                                    (35a) Br ethyl R5 = carboxy                                                   (36a) Br n-propyl R5 = carboxy                                                (37a) Br 3-methyl-n-butyl R5 = carboxy                                        (38a) Br 4-carboxy-n-butyl                                                    (39a) Br H R5 = carboxy                                                       (40a) Br ethyl R5 = amino                                                     (41a) Br n-propyl R5 = amino                                                  (42a) Br 3-methyl-n-butyl R5 = amino                                          (43a) Br ethyl R5 = 4-methylphenyl-                                              sulfonylamino                                                              (44a) Br n-propyl R5 = 4-methylphenyl-                                           sulfonylamino                                                              (45a) Br 3-methyl-n-butyl R5 = 4-methylphenyl-                                   sulfoylamino                                                               (46a) Br 2-methylbut-1-en-4-yl                                                (47a) Br 2-methylpent-2-en-5-yl                                               (48a) Br phenyl                                                               (49a) Br carboxy                                                              (55a) Br n-propyl R4 = R6 = carboxy                                         __________________________________________________________________________     .sup.2 Unless otherwise indicated, R3R6 = hydrogen.                      

As mentioned above, the compounds of Tables I and II have therapeuticactivity for example as antifungal agents, antibacterial agents, andantitumor agents. In addition, these compounds may be useful in the dyeindustry.

5. WORKING EXAMPLES

In this section, examples of the methods described above are providedfor illustration only and not by way of limitation. The reactants andstarting materials are either readily synthesized or purchased fromcommercial sources.

Example 1

2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone

1) Into a 250 ml round bottom flask, equipped with a magnetic stir bar,was placed 2-(3-methyl-n-butyl) indole (2.95 g), cesium carbonate (10.3g) bromanil (3.34 g), and acetonitrile (79 ml). The mixture was stirredat room temperature for 45 hours. Following dilution with 1 Nhydrochloric acid (250 ml), the crude mixture was extracted with ethylacetate (500 ml). The organic layer was washed with brine (200 ml) anddried with sodium sulfate. After removal of solvent under reducedpressure, the crude residue was filtered through a short plug of flashsilica, eluting with 20% ethyl acetate/hexane. The solvent was removedunder reduced pressure, and the residue was purified by flashchromatography (15% ethyl acetate/hexane) to yield2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (553 g,11%) as a blue crystalline solid.

Note: When the reaction was performed on 27.2 mg of starting indole, 95mg cesium carbonate, 31 mg bromanil and 0.72 ml acetonitrile, thereaction was complete after 1.5 hours, and the product yield was 28%.

2) To a stirred solution of2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (553mg), ethanol (9 ml), and tetrahydrofuran (9 ml) in a 100 ml round bottomflask equipped with a reflux condenser was added 2N aqueous potassiumhydroxide solution (9 ml). The mixture was heated at 85° C. for 13hours, followed by dilution with 1N aqueous sodium hydroxide solution(150 ml). The mixture was washed with 3:1 hexane/ethyl acetate (400 ml).After setting aside the aqueous layer, the organic layer was washed withanother portion of 1N aqueous sodium hydroxide solution (150 ml) andthen discarded. The basic aqueous layers were combined, acidified byadding 6N hydrochloric acid (60 ml), and extracted with ethyl acetate(300 ml). The organic layer was washed with brine (100 ml) and driedwith sodium sulfate. Removal of solvent afforded 345 mg (78%) of2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone as areddish-purple crystalline solid.

3) Preparation of 2-(3-methyl-n-butyl)-indole. Carbonyldiimidazole (65g) was mixed with 500 ml of dry dichloromethane in a 2-L round bottomflask and stirred magnetically. A solution of 4-methylvaleric acid in200 ml of dichloromethane was added dropwise over 45 minutes and themixture was stirred for another 1.25 hours. o-Toluidine (45 g) in 100 mlof dichloromethane was then added over about 20 minutes. After stirringfor 2 hours the mixture was washed with water and then the solvent wasstripped on a rotary evaporator. The residue was mixed with 150 ml ofmethanol and 75 ml of water and put in the freezer. Filtration of theprecipitate, dilution of the filtrate with water and refiltration of theprecipitate gave 75 g (94%) of vacuum driedN-(2-methylphenyl)-4-methylvaleramide which was used without furthertreatment in the next step.

The following procedure is cited in Bull. Soc. Chim. Fr. (37):190(1925). N-(2-methylphenyl)-4-methylvaleramide (20.5 g), sodium amide(90%) (11.0 g), and tetralin (100 ml) were mixed in a 500 ml roundbottom flask equipped with a magnetic stirrer and reflux condenser andheated at reflux for 2 hours. After cooling to room temperature, ethanol(10 ml) was added, followed by H₂ O (150 ml). The layers were separated,the organic layer was filtered through a pad of anhydrous magnesiumsulfate, and the solution was placed in a 200 ml round bottom flaskequipped with a 10-inch vacuum jacketed Vigreux column. Tetralin wasdistilled at 35-45° C./0.5 mm Hg. The residue was transferred to a 50 mlround bottom flask equipped with a 4-inch Vigreux column, anddistillation at 118-129° C./0.5 mm Hg provided2-(3-methyl-n-butyl)-indole 13.1 g (70%) as a slightly yellow solid.

Example 2

2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone(larger scale)

1) Into a 250 ml round bottom flask, equipped with a magnetic stir bar,was placed 2-(3-methyl-n-butyl)indole (30.0 g), cesium carbonate (62.6g) bromanil (27.2 g), and acetonitrile (64 ml). The mixture was stirredat room temperature for 20 hours. Following dilution with 1Nhydrochloric acid (500 ml), the crude mixture was extracted with ethylacetate (1 L). The organic layer was washed with brine (400 ml) anddried with sodium sulfate. The solvent was removed under reducedpressure, the residue was purified by flash chromatography (30% ethylacetate/hexane) to yield a 1:1 mixture of the desired product,2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone and theundesired by-product,2,6-dibromo-3,5-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (42.3 g,46% yield of desired product by HPLC) as a blue crystalline solid.

2) To a stirred solution of 1:1 mixture of2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone and2,6-dibromo-3,5-di[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (42.3 g),ethanol (166 ml), and tetrahydrofuran (166 ml) in a 1 L 3-necked roundbottom flask equipped with a reflux condenser was added 4N aqueouspotassium hydroxide solution (166 ml). The mixture was heated at 85° C.for 10 hours, followed by dilution with 1N hydrochloric acid (500 ml).The mixture was extracted with ethyl acetate (1 L). The organic layerwas washed with brine (250 ml) and dried with sodium sulfate. Removal ofsolvent afforded 35.1 g of crude (about 50% pure)2,5-Dihydroxy-3,6-di-[2-3-methyl-n-butyl)indol-3-yl]-1,4-quinone to bepurified by-HPLC.

In a separate experiment, purification of about 20 g of the crudeproduct (about 50% pure) by HPLC resulted in 5 g of2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone with apurity of 97%.

Example 3

2,5-Dihydroxy-3, 6-di-(2-n-butyl-indol-3-yl)-1, 4-quinone

This compound was synthesized in the same manner as Example 2 except thestarting indole was 2-n-butyl-indole.

Preparation of 2-n-butyl-indole

o-Toluidine (55 g) was mixed with 100 ml dry pyridine and 200 ml drytetrahydrofuran in a 1-L 3-necked round bottom flask fitted with aTrubore stirrer, thermometer and a dropping funnel, under nitrogen.Then, with cooling in a refrigerated bath, valeryl chloride (60.3 g) wasadded dropwise over 1 hour. The mixture was stirred for another hour atroom temperature and then poured onto 500 g ice and water. Theprecipitate was washed repeatedly with water on a Buchner funnel. Theprecipitate (88.9 g, 93%) was cyclized according to Verley and Bedure,1925, Bull. Soc. Chim. Fr. (37): 190 to afford 2-n-butyl indole (67.4 g,84%) as a very slightly yellow oil.

Example 4

2,5-Dihydroxy-3,6-di-[2-methyl-indol-3-yl]-1,4-quinone

This compound was synthesized in the same manner as Example 2 except thereaction time was 24 hours.

Example 5

2,5-Dihydroxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone

This compound may be synthesized as follows:

A mixture of 100 mg of 2,5-diacetoxy-3,6-dibromo-1,4-quinone, 180 mg of3-[2-(2-methylbut-2-en-4-yl)indole, prepared by the Fisher indolesynthesis, 10 ml of anhydrous dimethylforamide, and powdered potassiumcarbonate, is heated at 100° C. for 24 hours. The cooled mixture ispartitioned between ethyl acetate and water. The ethyl acetate layer isthen washed with brine, dried over sodium sulfate, filtered andconcentrated. The crude product is then purified on a medium pressureliquid chromatography column in a solvent mixture of dichloromethane andmethanol to provide 25 mg of2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone.2,5-Diacetoxy-3,6-di-[2(2-methylbut-2-en-4-5 yl)indol-3-yl]1,4-quinoneis then hydrolysed with 1 N aqueous sodium hydroxide solution inmethanol. Acidification of the above mixture produces the crude productafter filtration. Further crystallization in ethanol and water producesthe title compound.

Other suitably protected quinones such as3,6-dibromo-2,5-ditrimethylsiloxy-1,4-quinone,3,6-dibromo-2,5-di-(t-butyldimethylsiloxy-1,4-quinone,2,5-dibenzoxy-3,6-dibromo-1,4-quinone,3,6-dibromo-2,5-diisobutyroxy-1,4-quinone,2,5-dibenzyloxy-3,6-dibromo-1,4-quinone or2,5-diallyoxycarbonyloxy-3,6-dibromo-1,4-quinone which can be preparedfrom commercially available 2,4-dibromo-3,6-dihydroxy-1,4-quinone mayalso be used as starting materials. These protecting groups can beremoved by conventional deprotection methods such as diluted acid,potassium fluoride or palladium (0) complex or palladium on carbon withhydrogen or by methods described by Greene and Wuts (1991, "ProtectiveGroups In Organic Synthesis," John Wiley and Son). Other solvents suchas pyridine or dimethylsulfoxide (DMSO) may be used in place of dimethylformamide.

Example 6

2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone

2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone isprepared as in Example 5.

Example 7

2,5-Diacetoxy-3,6-di-[2(3-methyl-n-butyl)indol-3-yl]1,4-quinone

Hydrogenation of2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone inmethanol with 5% palladium on carbon under 1 atmosphere of hydrogenproduced the title compound.

Example 8

2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone

Base hydrolysis of2,5-diacetoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone asdescribed in Example 5 produced the title compound.

Under similar conditions as those described in Examples 5 to 8, thefollowing compounds are prepared using either2,5-dibromo-3,6-dihydroxy-1,4-quinone or 2,3,5,6-tetrabromoquinone asstarting materials:

Example 9

3,6-Di-[5-(bromo)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 10

3,6-Di-[2-(allyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 11

2,5-Dihydroxy-3,6-di-[2-(n-propyl)indol-3-yl]1,4-quinone

This compound was prepared under conditions similar to those describedin Examples 5 to 8.

Example 12

3,6-Di-[2-(aminocarbonyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 13

2,5-Diacetoxy-3,6-di-[2 (aminocarbonyl)indol-3-yl]-1,4-quinone

Example 14

3,6-Di-[2-allylindol-3-yl]-2,5-dibenzoyloxy-1,4-quinone

Example 15

2,5-Dihydroxy-3,6-di-[2-(cyano)indol-3-yl]1,4-quinone

Example 16

2,5-Dihydroxy-3,6-di-[4-(methoxycarbonyl)indol-3-yl]1,4-quinone

Example 17

2,5-Dihydroxy-3,6-di-[5,7-(dimethoxy)indol-3-yl]1,4-quinone

Example 18

2,5-Dihydroxy-3,6-di-[4,7-(dimethoxy)indol-3-yl]1,4-quinone

Example 19

2,5-Dihydroxy-3,6-di-[5-(nitro)indol-3-yl]1,4-quinone

Example 20

3,6-di-[4(4-chlorobenzoylamino)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 21

3,6-di-[2-(4-chlorophenyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 22

2,5-Dihydroxy-3,6-di-[2-(4-fluorophenyl)indol-3-yl]1,4-quinone

Example 23

2,5-Dihydroxy-3,6-di-[4,6-(dimethoxy)indol-3-yl]1,4-quinone

Example 24

2,5-Dihydroxy-3,6-di-[2-(5-hydroxy-6-methoxy)indol-3-yl]1,4-quinone

Example 25

2,5-Dihydroxy-3,6-di-[4-(cyano)indol-3-yl]1,4-quinone

Example 26

2,5-Dihydroxy-3,6-di-[5-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]1,4-quinone

Example 27

2,5-Dihydroxy-3,6-di-[2-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]1,4-quinone

Example 28

2,5-Dihydroxy-3,6-di-[2-(ethyl)indol-3-yl]1,4-quinone

This compound was prepared under conditions similar to those describedin Examples 5 to 8.

Example 29

3,6-di-[2-(5-bromo-6-nitro)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Example 30

2,5-Dimethoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone

Methylation of Example 5 with methyl iodide and potassium carbonate indimethylforamide followed by purification produces the title compound.This compound may also be prepared by heating2,5-dibromo-3,6-di[2-(2-methylbut-2-en-4-yl)indol-3-y]1,4-quinone inmethanol in the presence of powdered potassium carbonate.

Example 31

2,5-Dimethoxy-3,6-di-[2 (3-methyl-n-butyl)indol-3-yl]1,4-quinone

Hydrogenation of Example 30 under conditions as those in Example 3produced the title compound.

Example 32

Preparation of2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone

To a glass tube containing 2-(3-methyl-n-butyl)indole (400 mg), bromanil(431 mg) and potassium carbonate (703 mg) equipped with a magnetic stirbar, was added dimethylformamide (10 ml). The mixture was stirred atroom temperature for 40 hours. Following dilution with 1N hydrochloricacid (100 ml), the crude mixture was extracted with ethyl acetate (200ml). The organic layer was washed with brine (100 ml) and dried withsodium sulfate. After removal of solvent under reduced pressure, thecrude residue was filtered through a short plug of flash silica, elutingwith 30% ethyl acetate/hexane. The solvent was removed under reducedpressure, and the residue was purified by flash chromatography (15%ethyl acetate/hexane) to yield2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (40 mg,7%) as a blue crystalline solid.

To a stirred solution of2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-l,4-quinone (40 mg)in methanol (1.5 ml) was added 2N methanolic sodium hydroxide (0.251ml). The solution was stirred at room temperature for 24 hours, followedby dilution with water (50 ml). The product was extracted with ethylacetate (100 ml), washed with brine (50 ml) and dried with sodiumsulfate. Removal of solvent under reduced pressure provided2,5-methoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (30 mg,90%) as a yellow crystalline solid.

To a stirred solution of2,5-dimethoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (9 mg)in ethanol (2 ml) was added 1 N aqueous potassium hydroxide (1 ml). Themixture was heated at 85° C. for 3.5 hours, then diluted with 1 Nhydrochloric acid (25 ml). The product was extracted with ethyl acetate(50 ml), washed with brine (25 ml) and dried with sodium sulfate. Thesolvent was removed under reduced pressure to afford2,5-dihydroxy-3,6-di[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone (8 mg)as a reddish-brown stalline solid.

32a) Preparation of 2-(2-methyl-1-buten-4-yl)indole

To a stirred solution of 2-methylindole (1 g) in diethylether (76 ml)under nitrogen was added a 1.6 M solution of n-butyllithium in hexane(14.3 ml) slowly dropwise via syringe. Potassium tert-butoxide (1.711 g)was then added, producing a bright yellow mixture. After stirring atroom temperature under nitrogen for 50 minutes, the mixture was cooledto -78° C., whereupon 3-bromo-2-methylpropene (1.54 ml) was addeddropwise via syringe, giving a red-orange solution. The reaction mixturewas stirred at -78° C. for 2 hours, then quenched with water (10 ml).After warming to room temperature, water (150 ml) and 1 N hydrochloricacid (1 ml) was added to neutralize the reaction mixture. The mixturewas extracted with ethyl acetate (250 ml), and the organic layer waswashed with brine (100 ml) and dried with sodium sulfate. The solventwas removed under reduced pressure, and the crude residue was purifiedby flash chromatography (4% ethyl acetate/hexane) to afford2-(2-methyl-1-butene-4-yl)indole (664 mg. 47%) as a waxy yellow solid.

32b) This indole is preferably synthesized by the method of Example 1.However, this indole may also be prepared as follows:

Preparation of 2-(3-methyl-n-butyl)indole

Into a 3-necked round bottom flask under a blanket of nitrogen wasplaced 5% palladium catalyst on charcoal (771 mg). A solution of2-(2-methyl-1-buten-4-yl) indole (671 mg) in ethanol (36 ml) was addedto the flask, which was evacuated and charged with hydrogen twice. Themixture was stirred vigorously under hydrogen (1 atmosphere) for 2hours, followed by filtration through a pad of Celite. The solvent wasremoved under reduced pressure and the crude residue was purified byflash chromatography (3% ethyl acetate/hexane) to give2-(3-methyl-n-butyl)indole (400 mg, 59%) as a yellow crystalline solid.

Example 33

Preparation of 2,5-Dihydroxy-3,6-di-[2-(methyl)indol-3-yl]-1,4-quinone

This compound is preferably synthesized by the method of Example 2 using2-methylindole as the starting indole. However, this compound may alsobe prepared by the method of Example 32 using 2-methylindole as thestarting indole.

Example 34

Preparation of 3,6-Di-(2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone

This compound is preferably synthesized by the method of Example 2 using2-ethylindole as the starting indole. However, this compound may also beprepared by the method of Example 32 using 2-ethylindole as the startingindole.

Preparation of 2-ethylindole: Refer to 32a) using methyl iodide as thealkylating agent.

Example 35

Preparation of 3,6-Di-(2-butylindol-3-yl) 2,5-dihydroxy-1,4-quinone:This compound is preferably synthesized by the method of Example 2 using2-butylindole as the starting indole. Preparation of 2-butylindole:Refer to Example 3. However, this compound may also be prepared by themethod of Example 32 using 2-butylindole as the starting indole.

Preparation of 2-(but-1-en-4-yl)indole: Refer to 32a) using allylbromide as the alkylating agent. Preparation of 2-butylindole: Refer to32b) using 2-(but-1-en-4-yl)indole as the starting material.

Example 36

Preparation of3,6-Di-[2-(but-1-en-4-yl)indol-3-yl]2,5-dihydroxy-1,4-quinone

This compound is preferably synthesized by the method of Example 2 butmay also be prepared according to the method of Example 32 using2-(but-1-en-4-yl)indole as the starting indole.

Example 37

Preparation of2,5-Dihydroxy-3,6-di-[2-(4-methyl-n-pentyl)indol-3-yl]-1,4-quinone: Thiscompound is preferably synthesized by the method of Example 2 but mayalso be prepared according to Example 32 using 2-(4-methyl-n-pentyl)indole as the starting indole. Preparation of2-(2-methyl-2-penten-5-yl)indole: Refer to Example 1 using5-methylhexanoic acid as the starting acid. This indole may also beprepared according to Example 32a) using 4-bromo-2-methyl-2-butene asthe alkylating reagent. Preparation of 2-(4-methyl-n-pentyl)indole:Refer to 32b) using 2-(2-methyl-2-penten-5-yl)indole as the startingmaterial.

Example 38

Preparation of2,5-Dihydroxy-3,6-di-[2-(2-phenylethyl)indol-3-yl]-1,4-quinone: Thiscompound is preferably synthesized by the method of Example 2 but mayalso be prepared according to Example 32 using 2-(2-phenylethyl)indoleas the starting indole. Preparation of 2-(2-phenylethyl)indole: Refer toExample 3 using 3-phenylpropionyl chloride as the starting acidchloride. This indole may also be prepared according to Example 32a)using benzyl bromide as the alkylating agent.

Example 39

Preparation of2,5-Dihydroxy-6-(indol-3-yl)-3-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinon

This synthesis is achieved by treating 2-(3-methyl-n-butyl) indole with2 equivalents of bromanil in the presence of cesium carbonate indimethylformamide, followed by workup and purification similar toExample 32. The resultant mono-indolyl adduct is optionally treated with2 equivalents of indole under the same conditions as above to providethe bis-indolyl product.

Example 40

Preparation of3,6-Di-(5-carboxy-2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone: Refer toExample 32 using 5-carboxy-2-ethylindole as the starting indole.Preparation of 5-carboxy-2-ethylindole: Refer to Example 3 using methyl4-amino-3-methylbenzoate and propionyl chloride as the startingcompounds. The methyl ester is hydrolyzed upon workup of the cyclizationto give 5-carboxy-2-ethylindole. This synthesis may also be accomplishedbeginning with 5-chloro-2-methylindole, which is alkylated with methylindole. The product chloroindole is converted to its Grignard speciesand exposed to carbon dioxide to finish the synthesis.

Example 41

Preparation of3,6-Di-[5-carboxy-2-(n-propyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone:

Refer to Example 32 using 5-carboxy-2-propylindole as the startingindole. Preparation of 5-carboxy-2-propylindole: Refer to Example 1using methyl 4-amino-3-methyl-benzoate instead of o-toluidine or referto 40 using ethyl iodide as the alkylating agent.

Example 42

Preparation of3,6-Di-[5-carboxy-2-(3-methyl-n-butyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Refer to Example 32 using 5-carboxy-2-(3-methyl-n-butyl) indole as thestarting indole. Preparation of5-carboxy-2-(2-methyl-1-buten-4-yl)indole:

Refer to 40 using 3-bromo-2-methylpropene as the alkylating agent.Preparation of 5-carboxy-2-(3-methyl-n-butyl)indole: Refer to Example 1using methyl 4-amino-3-methyl-benzoate instead of o-toluidine, or referto Example 32b) using 5-carboxy-2-(2-methyl-l-buten-4-yl)indole as thestarting material.

Example 43

Preparation of3,6-Di-[2-(4-carboxy-n-butyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone:

Refer to Example 32 using 2-(4-carboxy-n-butyl)indole as the startingindole. Preparation of 2-(4-carboxy-3-buten-1-yl)indole:

Refer to 32(a) using 4-bromo-2-butenoic acid as the alkylating agent.Preparation of 2-(4-carboxy-n-butyl) indole: Refer to Example 3 usingmethyl adipyl chloride as the acid chloride. The methyl ester washydrolyzed in the cyclization workup to provide the productcarboxyindole. In the alternative, refer to Example 32b) using2-(4-carboxy-3-buten-1-yl)indole as the starting material.

Example 44

Preparation of3-[5-Carboxy-2-(3-methyl-n-butyl)indol-3-yl]-2,5-dihydroxy-6-(indol-3-yl)-1,4-quinone

Refer to Example 39 using 5-carboxy-2-(3-methyl-n-butyl) indole in thefirst step.

Example 45

Preparation of3,6-Di-(5-amino-2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone

Refer to Example 32 using 5-amino-2-ethylindole as the starting indole.Preparation of 5-amino-2-ethylindole: Refer to Example 3 using2-methyl-4-nitroaniline and propionyl chloride to give5-nitro-2-ethylindole, which is reduced to the desired amino compoundusing catalytic hydrogenation as in 32b.

In the alternative, this synthesis may be accomplished with a standardnitration of 2-ethylindole using sodium nitrate and sulfuric acidsimilar to that cited in Chem. Lett. (7): 1125-1128 (1991). Theresultant 5-nitro-2-ethylindole is reduced to the desired amino compoundusing catalytic hydrogenation as in 32b).

Example 46

Preparation of3,6-Di-[5-amino-2-(n-propyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Refer to Example 32 using 5-amino-2-(n-propyl)indole as the startingindole. Preparation of 5-amino-2-(n-propyl) indole: Refer to Example 45using butyryl chloride. In the alternative, refer to the synthesis citedin Chem. Lett. (7): 1125-1128 (1991) cited in Example 45 using2-n-propylindole.

Example 47

Preparation of3,6-Di-[5-amino-2-(3-methyl-n-butyl)indol-3-yl]2,5-dihydroxy-1,4-quinone

Refer to Example 32 using 5-amino-2-(3-methyl-n-butyl) indole as thestarting indole. Preparation of 5-amino-2-(3-methyl-n-butyl)indole:Refer to Example 1 using 2-methyl-4-nitroaniline instead of o-toluidine.The resultant 5-nitro-2-(3-methyl-n-butyl)-indole is reduced to thedesired amino compound as in 32b. The synthesis may also be accomplishedaccording to Example 45 using 2-(3-methyl-n-butyl)indole.

Example 48

Preparation of2,5-Diacetoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone

This synthesis was accomplished by treating2,5-hydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone withacetic anhydride in the presence of pyridine.

Example 49

Preparation of3,6-Di-[2-ethyl-5-(4-methylphenylsulfonylamino)indol-3-yl]-2,5-dihydroxy-1,4-quinone

Refer to Example 32 using 2-ethyl-5-(4-methylphenylsulfonylamino)indoleas the starting indole. Preparation of2-ethyl-5-(4-methylphenylsulfonylamino) indole: The above compound issynthesized by treating 5-amino-2-ethylindole with p-toluenesulfonylchloride in the presence of triethylamine.

Example 50

Preparation of2,5-Dihydroxy-3,6-di-[5-(4-methylphenylsulfonylamino)-2-(n-propyl)indol-3-yl]-1,4-quinone

Refer to Example 32 using5-(4-methylphenylsulfonylamino)-2-(n-propyl)indole as the startingindole. Preparation of5-(4-methylphenylsulfonylamino)-2-(n-propyl)indole: Refer to 49 using5-amino-2-propylindole.

Example 51

Preparation of2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamino)indol-3-yl]-1,4-quinone

Refer to Example 32 using2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamino)indole as thestarting indole. Preparation of2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamino)indole: Refer to 49using 5-amino-2-(3-methyl-n-butyl)indole.

Example 52

Preparation of2,5-Dihydroxy-3,6-di-[2-(2-methylbut-1-en-4-yl)indol-3-yl]-1,4-quinone

Refer to Example 32 using 2-(2-methylbut-1-en-4-yl) indole as thestarting indole.

Example 53

2,5-dihydroxy-2,6-di-[2-(2-methylpent-2-en-5-yl)-indol-3-yl]-1,4-quinone

Example 54

2,5-dihydroxy-3,6-di-(2-phenylindol-3-y-1)-1,4-quinone: Refer to Example2 using 2-phenylindole as the starting indole.

Example 55

2,5-dihydroxy-3,6-di-(2-carboxyindol-3-yl)-1,4-quinone

Example 56

Preparation of3-(2-carboxyindol-3-yl)-2,5-dihydroxy-6-(2-methylindol-3-yl)-1,4-quinone:

1) Into a 10 ml screw-cap glass tube was placed bromanil (1 g), cesiumcarbonate (2.3 g), ethyl indole-2-carboxylate (446 mg), and acetonitrile(2.36 ml). After stirring the mixture at room temperature for 3 hours,during which the mono-indolylquinone6-(2-ethylcarboxyindol-3-yl)-2,3,5-tribromo-1,4-quinone was formed,2-methylindole (464 mg) was added. The mixture was stirred at roomtemperature for 24 hours, after which 1 N hydrochloric acid (100 ml) wasadded. The aqueous layer was extracted with ethyl acetate (200 ml). Theorganic layer was washed with brine (100 ml) and dried with sodiumsulfate. Following removal of the solvent under reduced pressure, thecrude residue was purified by flash chromatography (30% ethylacetate/hexane) to provide2,5-dibromo-3-(2-ethylcarboxyindol-3-yl)-6-(2-methylindol-3-yl)-1,4-quinone(0.37 g) as a blue crystalline solid. Alternatively, themono-indolylquinone6-(2-ethylcarboxyindol-3-yl)-2,3,5-tribromo-1,4-quinone may isolatedseparately and used in further reactions with other indoles.

2) To a stirred solution of2,5-dibromo-3-(2-ethylcarboxyindol-3-yl)-6-(2-methylindol-3-yl)-1,4-quinone(0.37 g), ethanol (1.6 ml), and tetrahydrofuran (1.6 ml) was added 4 Naqueous potassium hydroxide solution (1.6 ml). The mixture was heated at85° C. for 10 hours, followed by dilution with 1 N hydrochloric acid (75ml). The mixture was extracted with ethyl acetate (150 ml). The organiclayer was washed with brine (75 ml) and dried with sodium sulfate.Removal of solvent afforded3-(2-carboxyindol-3-yl)-2,5-dihydroxy-6-(2-methylindol-3-yl)-1,4-quinone(0.258 g) as a reddish brown crystalline solid.

Example 57

Preparation of2,5-dihydroxy-6-(2-methylindol-3-yl)-3-(2-phenylindol-3-yl)-1,4-quinone.This compound was prepared according to the procedure of Example 56. Itdoesn't matter what order the two indoles are added in--the same productis obtained.

Example 58

Preparation of2,5-dihydroxy-6-[2-(3-methyl-n-butyl)indol-3-yl]-3-(2-phenylindol-3-yl)-1,4-quinone.This compound was prepared according to the procedure of Example 56. Asin Example 57, the order of addition of the two indoles doesn't matter.

Example 59

Preparation of6-[2-(n-butyl)-indol-3-yl]-3-(2-carboxyindol-3-yl)-2,5-dihydroxy-1,4-quinone.This compound was prepared according to the procedure of Example 56.

Example 60

Preparation of3-(2-carboxyindol-3-yl)-2,5-dihydroxy-6-[2-(n-propyl)-indol-3-yl]-1,4-quinone.This compound was prepared according to the procedure of Example 56.

Example 61

Preparation of3,6-di(6-carboxy-2-n-propylindol-3-yl)-2,5-dihydroxy-1,4-quinone: Referto Example 2 using 6-carboxy-2-n-propylindole as the starting indole.Preparation of 6-carboxy-2-n-propylindole: Refer to Example 3 usingmethyl 3-amino-4-methylbenzoate and butyryl chloride as the startingcompounds. The methyl ester was hydrolyzed upon workup of thecyclization to give 6-carboxy-2-n-propylindole.

6. FORMULATION AND USE OF INDOLYLCUINONES

The compounds disclosed herein have utility, inter alia, attherapeutically effective doses to treat or ameliorate cellproliferative disorders involving PTK/adaptor protein interactions. Thecompounds prepared according to the present invention may be tested by avariety of methods for determining the ability of the compounds toinhibit kinase activity or to disrupt PTK/adaptor protein complexes.

Any assay currently used for screening compounds that act on cellscontaining PTKs can be used. In general, such assays involve exposingcells that express the PTK to a test substance and either: (a) scoringphenotypic changes in the cell culture as compared to control cells thatwere not exposed to the test substance; or (b) biochemically analyzingcell lysates to assess the level and/or identity of tyrosinephosphorylated proteins.

A common technique involves incubating cells with ligand andradiolabeled phosphate, lysing the cells, separating cellular proteincomponents of the lysate using an SDS-polyacrylamide gel (SDS-PAGE)technique, in either one or two dimensions, and detecting the presenceof phosphorylated proteins by exposing X-ray film. In a similartechnique, the phosphorylated proteins are detected by immunoblottingtechniques, in which case the phosphate that is detected is notradiolabeled. Instead, the cellular components separated by SDS-PAGE aretransferred to a nitrocellulose membrane, where the presence ofphosphorylated tyrosines is detected using an antiphosphotyrosineantibody (anti-PY). The anti-PY can be detected by labeling it with aradioactive substance, or an enzyme, such as horseradish peroxidase. Afurther alternative involves detecting the anti-PY by reacting with asecond antibody which recognizes the anti-PY, this second antibody beinglabeled with either a radioactive moiety or an enzyme as previouslydescribed. Examples of these and similar techniques are described inHansen et al., 1993, Electrophoresis 14:112-126; Campbell et al. 1993,J. Biol. Chem. 268:7427-7434; Donato et al., 1992, Cell Growth and Diff.3:258-268; and Katagiri et al., 1993, J. Immunol. 150:585-593.

ELISA-type assays in microtitre plates can also be used to test purifiedsubstrates. See for example Peraldi et al., 1992, J. Biochem. 285:71-78; Schraag et al., 1993, Analytical Biochemistry 211:233-239;Cleavland, 1990, Analytical Biochemistry 190:249-253; Farley, 1992,Analytidal Biochemistry 203:151-157; and Lozaro, 1991, AnalyticalBiochemistry 192:257-261. Examples of assay methods are described inU.S. application Ser. No. 08/279,321, filed Jul. 22, 1994 and U.S.application Ser. No. 08/488,156 filed Jun. 7, 1995 which are herebyincorporated in their entireties by reference.

A variety of methods may be used to assay the ability of the compoundsprepared according to the invention to disrupt PTK/adaptor proteincomplexes. For example, in vitro complex formation may be assayed by,first, immobilizing one component, or a functional portion thereof, ofthe complex of interest to a solid support. Second, the immobilizedcomplex component may be exposed to a compound prepared according to thepresent invention, and to the second component, or a functional portionthereof, of the complex of interest. Third, it may be determined whetheror not the second component is still capable of forming a complex withthe immobilized component in the presence of the compound.

Additionally, in vivo complex formation may be assayed by utilizingco-immunoprecipitation techniques well known to those of skill in theart. Briefly, a cell line capable of forming a PTK/adaptor complex ofinterest may be exposed to one or more of the compounds preparedaccording to the present invention, and a cell lysate may be preparedfrom this exposed cell line. An antibody raised against one of thecomponents of the complex of interest may be added to the cell lysate,and subjected to standard immunoprecipitation techniques. In cases wherea complex is still formed, the immunoprecipitation will precipitate thecomplex, whereas in cases where the complex has been disrupted, only thecomplex component to which the antibody is raised will be precipitated.

The effect of a compound of the invention on the transformationcapability of the PTK/adaptor protein of interest may be directlyassayed. For example, one or more of the compounds prepared according tothe invention may be administered to a cell such as a fibroblast orhematopoietic cell capable of forming a PTK/adaptor complex which, inthe absence of a compound of the invention, would lead to the cell'stransformation (Muller, A. J. et al., 1991, Mol. Cell. Biol.11:1785-1792; McLaughlin, J. et al., 1987, Proc. Natl. Acad. Sci. USA84:6558-6562). The transformation state of the cell may then be measuredin vitro, by monitoring, for example, its ability to form colonies insoft agar (Lugo and Witte, 1989, Mol. Cell. Biol. 9:1263-1270; Gishizky,M. L. and Witte, O. N., 1992, Science 256:836-839). Alternatively, acell's transformation state may be monitored in vivo by determining itsability to form tumors in immunodeficient nude or severe combinedimmunodeficiency (SCID) mice (Sawyers, C. L. et al., 1992, Blood79:2089-2098). Further, the ability of the compounds prepared accordingto the present invention, to inhibit various tumor cell lines, such asfor example, melanoma, prostate, lung and mammary tumor cell linesestablished as SC xenografts can be examined.

Thus, the present invention also provides a method of amelioratingsymptoms of a cell proliferative disorder wherein the cell proliferativedisorder involves a protein tyrosine kinase polypeptide/adaptorpolypeptide complex, with an amount of a compound of either of theformulas I or XI, sufficient to disrupt protein tyrosine kinasepolypeptide/adaptor polypeptide complexes of the cell so that symptomsof the cell proliferative disorder are ameliorated.

The present invention also provides a method of ameliorating a cellproliferative disorder using a compound described herein, particularly acompound of the formula I, IV, V, VI, VIII, IX, X, XI, XII or XIII,wherein the cell proliferative disorder occurs in a mammal and thecompound contacts the cell within a mammal so that the symptoms of thecell proliferative disorder in the mammal are ameliorated. Thecompounds, i.e., indolylquinones, of the present invention may be usedalone or in combination with other drugs or therapies to treat cancer.

Cell proliferative disorders which are treatable according to themethods of the invention include BCR-ABL-associated cancers, gliomas,glioblastomas, melanomas, ovarian cancers, breast cancers, and prostatecancers.

Further, the present invention provides a method of amelioratingsymptoms of a cell proliferative disorder wherein the cell proliferativedisorder involves a. protein tyrosine kinase polypeptide/adaptorpolypeptide complex, which involves contacting a cell capable of formingthe protein tyrosine kinase polypeptide/adaptor polypeptide complex withan amount of a pharmaceutical composition comprising a compound of anyone of the formulas I, IV, V, VI, VIII, IX, X, XI, XII or XIII,sufficient to disrupt protein tyrosine kinase polypeptide/adaptorpolypeptide complexes of the cell so that symptoms of the cellproliferative disorder are ameliorated.

Further, the compounds prepared according to the present invention maybe formulated into compositions comprising other drugs or pharmaceuticalagents. In one embodiment, the pharmaceutical compositions of compoundsprepared according to the present invention also comprise additionalcancer treatment agents. For example, the compounds prepared accordingto the present invention may be formulated into pharmaceuticalcompositions in a conventional manner using one or more physiologicallyacceptable carriers or excipients. The compounds and theirphysiologically acceptable salts and solvates may also be formulated foradministration by inhalation (either through the mouth or the nose) ororal, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents and other pharmaceutical agents asappropriate.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. Such formulations may also comprise otherpharmaceutical agents as appropriate. In the case of a pressurizedaerosol the dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of e.g. gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound and a suitable powder base such as lactose orstarch.

Compounds prepared according to the present method may also beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds that can be prepared according to the present methods andtheir methods of use can also be found in United States patentapplication Ser. Nos. 08/476,136, filed Jun. 7, 1995 and 08/658,337,filed Jun. 5, 1996, each of which is hereby incorporated by reference.

EXAMPLE--In Vivo Activity of Compound #1

The following example illustrates the use of the compounds of theinvention in an in vivo model of tumor growth.

Materials and Methods

Female athymic mice (BALB/c, nu/nu) were obtained from SimonsenLaboratories (Gilroy, Calif.). All animals were maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They received sterile rodent chow and water ad libitum. A cell lineestablished from a human epidermoid carcinoma (A431, ATCC CRL 1555) wasgrown in DMEM with 10% FBS and 2 mM GLN. All cell culture media,glutamine, and fetal bovine serum were purchased from Gibco LifeTechnologies (Grand Island, N.Y.) unless otherwise specified. All cellswere grown in a humid atmosphere of 90-95% air and 5-10% CO2 at 37° C.All cell lines were routinely subcultured twice a week and were negativefor mycoplasma as determined by teh Mycotect method (Gibco). Cells wereharvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at450×g for 10 min. Pellets were resuspended in sterile PBS or media(without FBS) to a particular concentration and the cells were implantedinto the hindflank of the nice (8-1 mice per group). Tumor growth wasmeasured over 3 to 6 weeks using venier calipers. Tumor volumes werecalculated as a product of length ×width ×height unless otherwiseindicated. P values were calculated using the Students' t-test. Compound1 (from Example 1 and Table I) in 100 μL excipient (VPD:D5W, 1:1 (VPD-12% w/v polysorbate 80, 0.55% citric acid (anhydrous), 35% w/vpolyenthlene glycol (MW=300 daltons) and 26.3% v/v 190 proof ethanoldiluted 1:22 in 5% dextrose in water (D5W) was delivered by IP injectionat different concentrations. Control animals received VPD:D5W alone.Animals were dosed daily on days 1-5 (high dose), days 1-9 (mid-dose) orthroughout the study (low dose).

Results

The results are shown below as a percent tumor reduction compared tocontrols. Administration of compound I inhibited tumor growth in a dosedependent manner. Tumor growth remained inhibited even after cessationof treatment with the compound.

    ______________________________________                                        Dosage                                                                          mg/kg/day Day % inhibition p-value                                          ______________________________________                                        2.5       7           11        ns                                               18  0 ns                                                                     5 7 30 <0.01                                                                   18 38 <0.01                                                                  7.5 7 55 <0.01                                                                 18 50 <0.01                                                                ______________________________________                                    

The present invention is not to be limited in scope by the specificexamples or embodiments described herein. These examples are, thus, notto be construed as limiting the scope of the invention in any way.Indeed, various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the amended claims.

What is claimed is:
 1. A compound of the formula: ##STR28## or apharmaceutically acceptable salt thereof, wherein: R₁ and R₂ are eachindividually Br, Cl, F, I, H, OH or --OCOR, wherein R is lower alkyl,aryl or alkylaryl;R₃₀ is Br, Cl, F, I, OH or --OCOR, wherein R is loweralkyl, aryl or alkylaryl; R₁ " is H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂-C₇ alkynyl, arylalkyl or aryl; R₃ to R₇ are each independentlyhydrogen, branched or unbranched C₁ -C_(n) alkyl, alkylcarboxy, C₂-C_(m) alkenyl, alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy,hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo, trihalomethyl, amido,carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or2-metlhylbut-2-en-4-yl, wherein n is an integer from 0 to 12 and m is aninteger of 2 to
 12. 2. A compound of the formula: ##STR29## or apharmaceutically acceptable salt thereof, wherein: R₂ and R₃₀ are eachindividually Br, Cl, F, I, H, OH or --OCOR, wherein R is lower alkyl,aryl or alkylaryl;R₁ is Br, Cl, F, I, OH or --OCOR, wherein R is loweralkyl, aryl or alkylaryl; R₁ " is H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂-C₇ alkynyl, arylalkyl or aryl; and R₃ to R₇ are each independentlyhydrogen, branched or unbranched C₁ -C_(n) alkyl, alkylcarboxy, C₂-C_(m) alkenyl, alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy,hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo, trihalomethyl, amido,carboxamido, carboxy, sulfonlyl, sulfonamido, amino, mercapto, or2-methylbut-2-en-4-yl, wherein n is an integer from 0 to 12 and m is aninteger of 2 to
 12. 3. A compound of the formula: ##STR30## or apharmaceutically acceptable salt thereof, wherein: R₁ and R₃₀ are eachindividually Br, Cl, F, I, H, OH or --OCOR, wherein R is lower alkyl,aryl or alkylaryl;R₂ is Br, Cl, F, I, OH or --OCOR, wherein R is loweralkyl, aryl or alkylaryl; R₁ " is H, C₁ -C₇ alkyl, C₂ -C₇ alkenyl, C₂-C₇ alkynyl, arylalkyl or aryl; and R₃ to R₇ are each independentlyhydrogen, branched or unbranched C₁ -C_(n) alkyl, alkylcarboxy, C₂-C_(m) alkenyl, alkynyl, alkenylcarboxy, aryl, alkylaryl, hydroxy,hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo, trihalomethyl, amido,carboxamido, carboxy, sulfonyl, sulfonamido, amino, mercapto, or2-methylbut-2-en-4-yl, wherein n is an integer from 0 to 12 and m is aninteger of 2 to
 12. 4. A compound of the formula: ##STR31## or apharmaceutically acceptable salt thereof, wherein: R₁, R₂ and R₃₀ areeach individually Br, Cl, F, I, H, OH or --OCOR, wherein R is loweralkyl, aryl or alkylaryl;R₁ " is C₂ -C₇ alkynyl, arylalkyl or aryl; R₃to R₇ are each independently hydrogen, branched or unbranched C₁ -C_(n)alkyl, alkylcarboxy, C₂ -C_(m) alkenyl, alkynyl, alkenylcarboxy, aryl,alkylaryl, hydroxy, hydroxyalkyl, C₁ -C_(n) alkoxy, nitro, halo,trihalomethyl, amido, carboxamido, carboxy, sulfonyl, sulfonamido,amino, mercapto, or 2-methylbut-2-en-4-yl, wherein n is an integer from0 to 12 and m is an integer of 2 to
 12. 5. A compound selected from thegroup consisting of compounds of the formula: ##STR32## a) wherein R₁,R₂ and R₃₀ are Br, R₇ is 3-methyl-n-butyl, and R₁ " and R₃ -R₆ are H;b)wherein R₁, R₂ and R₃₀ are Br, R₇ is n-butyl, and R₁ " and R₃ -R₆ are H;c) wherein R₁, R₂ and R₃₀ are Br, R₇ is methyl, and R₁ " and R₃ -R₆ areH; d) wherein R₁, R₂ and R₃₀ are Br, R₇ is 2-methylbut-2-en-4yl, and R₁" and R₃ -R₆ are H; e) wherein R₁, R₂ R₅ and R₃₀ are Br, and R₁ ", R₃,R₄, R₆ and R₇ are H; f) wherein R₁, R₂ and R₃₀ are Br, R₇ is allyl, andR₁ " and R₃ -R₆ are H; g) wherein R₁, R₂ and R₃₀ are Br, R₇ is n-propyl,and R₁ " and R₃ -R₆ are H; h) wherein R₁, R₂ and R₃₀, are Br, R₇ isaminocabonyl, and R₁ " and R₃ -R₆ are II; i) wherein R₁, R₂ and R₃₀ areBr, R₇ is cyano, and R₁ " and R₃ -R₆ are H: j) wherein R₁, R₂ and R₃₀are Br, R₆ is methoxycarbonyl, and R₁ ", R₃ -R₆, and R₇ are H; k)wherein R₁, R₂ and R₃₀ are Br, R₃ and R₆ are methoxy, and R₁ ", R₄, R₅and R₇ are H; l) wherein R₁, R₂ and R₃₀ are Br, R₅ is nitro, and R₁ ",R₃, R₄, R₆ and R₇ are H; m) wherein R₁, R₂ and R₃₀ are Br, R₆ is4-chlorobenzoylamino, and R₁ ", R₃ R₅ and R₇ are H; n) wherein R₁, R₂and R₃₀ are Br, R₇ is 4-chlorophenyl, and R₁ " and R₃ -R₆ are H; o)wherein R₁, R₂ and R₃₀ are Br, R₇ is 4-fluorophenyl, and R₁ " and R₃ -R₆are H; p) wherein R₁, R₂ and R₃₀ are Br, R₁ and R₆ are methoxy, and R₁", R₃, R₅ and R₇ are H; q) wherein R₁, R₂ and R₃₀ are Br, R₄ and R₅ aremethoxy, and R₁ ", R₃, R₆ and R₇ are H; r) wherein R₁, R₂ and R₃₀ areBr, R₆ is cyano, and R₁ ", R₃ -R₅ and R₇ are H; s) wherein R₁, R₂ andR₃₀ are Br, R₅ is trifluoromethylphenylaminocarbonyl and R₁ ", R₃, R₄,R₆ and R₇ are H; t) wherein R₁, R₂ and R₃₀ are Br, R₇ is4-trifluoromethylphenylaminocarbonyl, and R₁ " and R₃ R₆ are H; u)wherein R₁, R₂ and R₃₀ are Br, R₇ is ethyl, and R₁ " and R₃ -R₆ are H;v) wherein R₁, R₂ R₅ and R₃₀ are Br, R₄ is NO₂, and R₁ ", R₃, R₆ and R₇are H; w) wherein R₁, R₂ and R₃₀ are Br, R₇ is but-1-en-4-yl, and R₁ "and R₃ -R₆ are H; x) wherein R₁, R₂ and R₃₀ are Br, R₇ is4-methyl-n-pentyl, and R₁ " and R₃ -R₆ are H; y) wherein R₁, R₂ and R₃₀are Br, R₇ is 2-phenylethyl, and R₁ " and R₃ -R₆ are H; z) wherein R₁,R₂ and R₃₀ are Br, and R₁ " and R₃ -R₇ are H; aa) wherein R₁, R₂ and R₃₀are Br, R₇ is carboxy, R₇ is ethyl, and R₁ ", R₃, R₄ and R₆ are H; ab)wherein R₁, R₂ and R₃₀ are Br, R₅ is carboxy, R₇ is n-propyl, and R₁ ",R₃, R₄ and R₆ are H; ac) wherein R₁, R₂ and R₃₀ are Br, R₅ is carboxy,R₇ is 3-methyl-n-butyl, and R₁ ", R₃, R₄ and R₆ are H; ad) wherein R₁,R₂ and R₃₀ are Br, R₇ is 4-carboxy-n-butyl, and R₁ " and R₃ -R₆ are H;ae) wherein R₁, R₂ and R₃₀ are Br, R₅ is carboxy, and R₁ ", R₃, R₄, R₆and R₇ are H; af) wherein R₁, R₂ and R₃₀ are Br, R₅ is amino, R₇ isethyl, and R₁ ", R₃, R₄ and R₆ are H; ag) wherein R₁, R₂ and R₃₀ are Br,R₅ is amino, R₇ is n propyl, and R₁ ", R₃, R₄ and R₆ are H; ah) whereinR₁, R₂ and R₃₀ are Br, R₅ is amino, R₇ is 3-methyl-n-butyl, and R₁ ",R₃, R₄ and R₆ are H; ai) wherein R₁, R₂ and R₃₀ are Br, R₅ is4-methylphenylsulfonylamino, R₇ is ethyl, and R₁ ", R₃, R₄ and R₆ are H;aj) wherein R₁, R₂ and R₃₀ are Br, R₅ is 4-methylphenylsulfonylamino, R₇is n-propyl, and R₁ ", R₃, R₄ and R₆ are II; ak) wherein R₁, R₂ and R₃₀are Br, R₅ is 4-methylphenylsulfonylamino, R₇ is 3-methyl-n-butyl, andR₁ ", R₃, R₄ and R₆ are II; al) wherein R₁, R₂ and R₃₀ are Br, R₇ is2-methylbut-1-en-4-yl, and R₁ " and R₃ -R₆ are H; am) wherein R₁, R₂ andR₃₀ are Br, R₇ is 2-methylpent-2-en-5-yl, and R₁ " and R₃ -R₆ are H; an)wherein R₁, R₂ and R₃₀ are Br, R₇ is phenyl, and R₁ " and R₃ -R₆ are H;ao) wherein R₁, R₂ and R₃₀ are Br, R₇ is carboxy, and R₁ " and R₃ -R₆are H; and ap) wherein R₁, R₂ and R₃₀ are Br, R₄ and R₆ are carboxy, R₇is n-propyl, and R₁ ", R₃ and R₆ are H;or a pharmacentically acceptablesalt thereof.
 6. A pharmaceutical composition which comprises atherapeutically effective amount of a compound of claim 1, 2, 3 or 4 anda pharmaceutically acceptable carrier or excipient.